1. Field of the Invention
The present invention relates to the fields of Surface Enhanced Raman Scattering (SERS) using a single and arrays of nanoplasmonic resonators for detection of enzymatic activity. The present invention relates specifically to the detection of protease activity, such as the Prostate Specific Antigen (PSA) and Proteolytically Active PSA for diagnostic applications in prostate cancer.
2. Related Art
Originally developed in 1928, Raman spectroscopy has been used extensively to characterize molecular properties (Kazuo Nakamoto John R. Ferraro, Chris W. Brown, Introductory Raman Spectroscopy, 2nd ed. (Elsevier Science, 2003). Surface-Enhanced Raman Spectroscopy (SERS) increases the Raman signal significantly (C. R. Yonzon, C. L. Haynes, X. Zhang et al., Anal Chem 76 (1), 78 (2004); A. E. Grow, L. L. Wood, J. L. Claycomb et al., J Microbiol Methods 53 (2), 221 (2003); K. Nithipatikom, M. J. McCoy, S. R. Hawi et al., Anal Biochem 322 (2), 198 (2003); J. B. Jackson and N. J. Halas, P Natl Acad Sci USA 101 (52), 17930 (2004)) through enhanced electromagnetic fields in close proximity to a surface. SERS measurements performed on rough metal surface or dispersed metal nanoparticle aggregates have shown the highest Raman enhancement factors up to 1014 for detection down to single molecule level (Katrin Kneipp, Harald Kneipp, Irving Itzkan et al., Current Science (Bangalore) 77 (7), 915 (1999); S. Nie and S. R. Emory, Science 275 (5303), 1102 (1997)) but these measurement often suffer from poor reproducibility (M. Moskovits, Reviews of Modern Physics 57 (3), 783 (1985)). To improve the reproducibility, other methods including self-assembly of metallic colloidal nano-particles (R. G. Freeman, K. C. Grabar, K. J. Allison et al., Science 267 (5204), 1629 (1995)), nanosphere lithography (NSL) and metal film over nanosphere (MFON) (C. L. Haynes and R. P. Van Duyne, Journal of Physical Chemistry B 107 (30), 7426 (2003)), electrochemical roughening of polished gold substrate (J. M. Sylvia, J. A. Janni, J. D. Klein et al., Analytical Chemistry 72 (23), 5834 (2000)), and periodic structured metallic substrate using electron-beam lithography (M. Kahl, E. Voges, S. Kostrewa et al., Sensors and Actuators B-Chemical 51 (1-3), 285 (1998)), have been developed to fabricate SERS substrate consisting homogeneous features over large area with reproducible enhancement factors up to 108. Although these efforts lead to successful utilization of SERS analysis in many promising applications including gene and protein discrimination (Y. C. Cao, R. C. Jin, J. M. Nam et al., J Am Chem Soc 125 (48), 14676 (2003); Y. W. C. Cao, R. C. Jin, and C. A. Mirkin, Science 297 (5586), 1536 (2002); T. Vo-Dinh, F. Yan, and M. B. Wabuyele, Journal of Raman Spectroscopy 36 (6-7), 640 (2005)), bio-warfare agents detection (D. A. Stuart, K. B. Biggs, and R. P. Van Duyne, Analyst 131 (4), 568 (2006)) and real-time glucose monitoring (O. Lyandres, N. C. Shah, C. R. Yonzon et al., Analytical Chemistry 77 (19), 6134 (2005)), lacking of the ability to fabricate SERS hot-spots at specific location limits application for very small sample volume.
To overcome such limit, we recently developed tunable nanoplasmonic resonators (NPRs), consisting of thin SiO2 layer sandwiched between metallic nano-disks described in Durant S. Su K, Steel M. J., Xiong Y. Sun C., Zhang X, Journal of Physical Chemistry B 110 (9), 3964 (2006) hereby incorporated by reference. The resonance frequency can be precisely tuned by varying the dielectric layer thickness and aspect-ratio of the NPRs. Individual NPRs can enhance the Raman intensity by a factor of 6.1×1010; among the largest values obtained for a single SERS substrate or nanoparticle. Fabricated using well established nanolithography processes, the NPR-based method enables producing SERS hot-spots at desired location in a much smaller dimension reproducibly, allowing multiplexed high throughput detection and lab-on-chip applications.
Prostate cancer biomarker Prostate Specific Antigen (PSA), a kallikrein (hK) family serine protease (S. R. Denmeade and J. T. Isaacs, BJU Int 93 Suppl 1, 10 (2004); J. A. Clements, N. M. Willemsen, S. A. Myers et al., Crit Rev Clin Lab Sci 41 (3), 265 (2004)), is used as a model protease in the present application. The commonly used prostate-specific antigen (PSA) blood test has being widely used for early diagnosis and management of prostate cancer, the leading male cancer (H. Gronberg, Lancet 361 (9360), 859 (2003); S. R. Denmeade and J. T. Isaacs, Nat Rev Cancer 2 (5), 389 (2002)). However, serum PSA concentrations reflect the presence of benign prostatic hyperplasia (BPH) more often than cancer (A. Caplan and A. Kratz, Am J Clin Pathol 117 Suppl, S104 (2002); E. I. Canto, S. F. Shariat, and K. M. Slawin, Curr Urol Rep 5 (3), 203 (2004)). The lack of specificity causes a high false-positive rate and often leads to costly prostate needle biopsies for diagnosis and post-biopsy complications as well as considerable anxiety (M. B. Gretzer and A. W. Partin, Urol Clin North Am 30 (4), 677 (2003); A. Haese, M. Graefen, H. Huland et al., Curr Urol Rep 5 (3), 231 (2004)). Recent research has identified a family of highly specific peptides that can be cleaved by paPSA isoform in xenografts models (S. R. Denmeade, C. M. Jakobsen, S. Janssen et al., J Natl Cancer Inst 95 (13), 990 (2003)) and human samples (P. Wu, U. H. Stenman, M. Pakkala et al., Prostate 58 (4), 345 (2004); P. Wu, L. Zhu, U. H. Stenman et al., Clin Chem 50 (1), 125 (2004)) thus, measurement of paPSA protease activity from in vivo samples is possible and would be potentially valuable as a more specific screening agent for prostate cancer and in detection of recurrent disease. However, reported results based on immunopeptidemetric assays (IMPA) exhibit low fluorescence signal-to-noise ratios, preventing reliable measurements at lower concentrations in the clinically important range of 60-300 pM (P. Wu, U. H. Stenman, M. Pakkala et al., Prostate 58 (4), 345 (2004); P. Wu, L. Zhu, U. H. Stenman et al., Clin Chem 50 (1), 125 (2004)). In addition, there is usually a limited number of prostate cancer cells (<1000) isolated from fine needle biopsy or circulating cell capture. No commercial method exists that can perform a paPSA protease activity assay on a small number of cells for clinical staging. Therefore, one goal of the present invention is to provide a method that allows specific and sensitive measurements of paPSA for prostate cancer detection in a very small sample volume.